Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-05-21
2002-04-02
Huff, Sheela (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S004000, C435S007210, C435S007200, C435S007230, C530S350000, C530S387100, C530S387700
Reexamination Certificate
active
06365357
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to uses of immunological reagents specific for a human transmembrane efflux pump protein (P-glycoprotein). Specifically, the invention relates to uses of such immunological reagents that specifically recognize P-glycoprotein that is in a biochemical conformation adopted in the presence of certain cytotoxic, lipophilic drugs that are substrates for P-glycoprotein, in the presence of cellular ATP depleting agents, and by certain mutant embodiments of Pgp. In particular, the invention provides methods of using such immunological reagents for anticancer drug screening and development.
2. Background of the Invention
Many human cancers express intrinsically or develop spontaneously resistance to several classes of anticancer drugs, each with a different structure and different mechanism of action. This phenomenon, which can be mimicked in cultured mammalian cells selected for resistance to certain plant alkaloids or antitumor antibiotics such as colchicine, vinblastine and doxorubicin (formerly known as Adriamycin), is generally referred to as multidrug resistance (“MDR”; see Roninson (ed)., 1991
, Molecular and Cellular Biology of Multidrug Resistance in Tumor Cells
, Plenum Press, N.Y., 1991; Gottesman et al., 1991, in
Biochemical Bases for Multidrug Resistance in Cancer
, Academic Press, N.Y., Chapter 11 for reviews). The MDR phenotype presents a major obstacle to successful cancer chemotherapy in human patients.
MDR frequently appears to result from decreased intracellular accumulation of anticancer drugs as a consequence of increased drug efflux related to alterations at the cellular plasma membrane. When mutant cell lines having the MDR phenotype are isolated, they are found to express an ATP-dependent non-specific molecular “pump” protein (generally known as P-glycoprotein) that is located in the plasma membrane and keeps the intracellular accumulation of an anti-cancer drug low enough to evoke the drug-resistance phenotype. This protein (which has been determined to be the gene product of the MDR1 gene in humans) facilitates active (i.e., energy-dependent) drug efflux from the cell, against a concentration gradient of (generally) lipophilic compounds, including many cytotoxic drugs.
The gene encoding P-glycoprotein (which is also known as gp170-180 and the multidrug transporter) has been cloned from cultured human cells by Roninson et al. (see U.S. Pat. No. 5,206,352, issued Apr. 27, 1993), and is generally referred to as MDR1. The proteinproduct of the MDR1 gene, most generally known as P-glycoprotein (“Pgp”), is a 170-180 kilodalton (kDa) transmembrane protein having the aforementioned energy-dependent efflux pump activity.
Molecular analysis of the MDR1 gene indicates that Pgp consists of 1280 amino acids distributed between two homologous halves (having 43% sequence identity of amino acid residues), each half of the molecule comprising six hydrophobic transmembrane domains and an ATP binding site within a cytoplasmic loop. Only about 8% of the molecule is extracellular, and carbohydrate moieties (approximately 30 kDa) are bound to sites in this region (Chen et al., 1986
, Cell
47: 381-387).
Expression of Pgp on the cell surface is sufficient to render cells resistant to many (but not all) cytotoxic drugs, including many anti-cancer agents. Pgp-mediated MDR appears to be an important clinical component of drug resistance in tumors of different types, and MDR 1 gene expression correlates with resistance to chemotherapy in different types of cancer.
Pgp is also constitutively expressed in many normal cells and tissues (see Cordon-Cardo et al., 1990
, J. Histochem. Cytochem
. 38: 1277; and Thiebaut et al., 1987
, Proc. Natl. Acad. Sci. USA
84: 7735 for reviews). In hematopoietic cells, Neyfakh et al. (1989
, Exp. Cancer Res
. 185: 496) have shown that certain subsets of human and murine lymphocytes efflux Rh123, a fluorescent dye that is a Pgp substrate, and this process can be blocked by small molecule inhibitors of Pgp. It has been demonstrated more recently that Pgp is expressed on the cell-surface membranes of pluripotent stem cells, NK cells, CD4- and CD8-positive T lymphocytes, and B lymphocytes (Chaudhary et al., 1992
, Blood
80: 2735; Drach et al., 1992
, Blood
80: 2729; Kimecki et al., 1994
, Blood
83: 2451; Chaudhary et al., 1991
, Cell
66: 85). Pgp expression on the cell surface membranes of different subsets of human lymphocytes has been extensively documented (Coon et al., 1991
, Human Immunol
. 32: 134; Tiirikainen et al., 1992
, Ann. Hematol
. 65: 124; Schluesener et al., 1992
, Immunopharmacology
23: 37; Gupta et al., 1993
, J. Clin. Immunol
. 13: 289). Although recent studies suggest that Pgp plays a role in normal physiological functions of immune cells (Witkowski et al., 1994
, J. Immunol
. 153: 658; Kobayashi et al., 1994
, Biochem. Pharmacol
. 48: 1641; Raghu et al., 1996
, Exp. Hematol
. 24: 1030-1036), the physiological role of Pgp in normal immune cells has remained unclear to date.
Once the central role in MDR played by Pgp was uncovered, agents with a potential for reversing MDR phenotypes were developed that target Pgp. Several classes of drugs, including calcium channel blockers (e.g., verapamil), immunosuppresants (such as cyclosporines and steroid hormones), calmodulin inhibitors, and other compounds, were found (often fortuitously) to enhance the intracellular accumulation and cytotoxic action of Pgp-transported drugs (Ford et al., 1990
, Pharm. Rev
. 42: 155). Many of these agents were found to inhibit either drug binding or drug transport by Pgp (Akiyamaetal., 1988
, Molec. Pharm
. 33: 144; Horio et al., 1988
, Proc. Natl. Acad. Sci. USA
84: 3580). Some of these agents themselves were found to bind to and be effluxed by Pgp, suggesting that their enhancing effects on the cytotoxicity of Pgp substrates are due, at least in part, to competition for drug binding sites on this protein (Cornwell et al., 1986
, J. Bio. Chem
. 261: 7921; Tamai, 1990
, J. Biochem. Molec. Biol
. 265: 16509).
Many of these agents, however, also have strong, deleterious side effects at physiologically-achievable concentrations. These systemic side effects severely limit the clinical use of these agents as specific inhibitors of Pgp or for negative selection against Pgp-expressing tumor cells. Most of the known MDR-reversing drugs used in clinical trials have major side effects unrelated to inhibition of Pgp, such as calcium channel blockage (verapamil) or immunosuppression (cyclosporines and steroids). Similarly, targeting of cytotoxic drugs to Pgp-expressing cells is capable of compromising normal tissue function in normal cells (such as kidney, liver, colonic epithelium, etc.) that normally express Pgp. These drawbacks restrict the clinically-achievable dose of such agents and ultimately, their usefulness.
Immunological reagents also provide a means for specifically inhibiting drug efflux mediated by Pgp. Monoclonal antibodies specific for Pgp are known in the art.
Hamada et al., 1986
, Proc. Natl. Acad. Sci. USA
83: 7785 disclose the mAbs MRK-16 and MRK-17, produced by immunizing mice with doxorubicin-resistant K-562 human leukemia cells. MRK-16 mAb was also reported to modulate vincristine and actinomycin D transport in resistant cells, and MRK-17 was shown to specifically inhibit growth of resistant cells with these drugs.
Meyers et al., 1987
, Cancer Res
. 49: 3209 disclose mAbs HYB-241 and HYB-612, which recognize an external epitope of Pgp.
O'Brien et al., 1989
, Proc. Amer. Assoc. Cancer Res
. 30: Abs 2114 disclose that mAbs HYB-241 and HYB-612 increased the accumulation of vincristine and actinomycin D in tumor cells and increased the cytotoxicity of combinations of these drugs with verapamil.
Tsuruo et al., 1989
, Jpn. J. Cancer Res
. 80: 627 reported that treatment of athymic mice that had been previously inoculated with drug resistant human ovarian cancer cells with the mAb MRK16 caused regression of established subcutaneous tumors.
Hamada et al., 1990
, Canc
Fruehauf John
Mechetner Eugene
Harris Alana M.
Huff Sheela
McDonnell & Boehnen Hulbert & Berghoff
Onotech, Inc.
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